The field of this invention is fluorescent detection in microfluidic arrays.
1. Background
The combination of combinatorial chemistry, sequencing of the genomes of new species and relationships between genotype and physical and biological traits has greatly expanded the need to perform determinations of different events The multiplicity of new compounds that can be prepared using various forms of combinatorial chemistry and the numerous targets involving wild-type and mutated genes, had extraordinarily increased the number of determinations of interest in developing compounds having biological activity. These compounds include drugs, biocides, pesticide resistance, disease organism resistance and the like. In addition, the interest in discriminating between different genomes, relating specific mutations to phenotypes, defining susceptibilities to various environmental effects in relation to single nucleotide polymorphisms, and identifying the genomes of organisms to provide better defenses against the organisms has expanded the need for rapid inexpensive devices and methodologies for carrying out these and other determinations.
Recently, microfluidic arrays have been developed which allow for a multiplicity of reservoirs and channels to be associated with a small card or chip, where by using high voltages, various operations can be performed. The arrays provide for individual networks, which exist in combination on a single chip, so that a plurality of determinations may be performed concurrently and/or consecutively By having channels that have cross-sections in the range of about 500 to 5000 xcexc2, operations can be carried out with very small volumes In addition, by having very sensitive detection systems, very low concentration of a detectable label may be employed This allows for the use of very small samples and small amounts of reagents, which have become increasingly more sophisticated and expensive Micofluidic arrays offer the promise of more rapid throughput, increasingly smaller times to a determination and increasingly smaller amounts of sample and reagents being required
The use of microfluidic arrays, however, is not without its challenges. The microfluidic arrays are desirably made in molded plastic, so as to provide a reduced cost of the chip. By molding the chip and providing for ridges on a mold to form the channels, the channels may not run true and may be displaced from their proper positions, as well as being slightly curved rather than perfectly straight. In addition, the plastic frequently autofluoresces. Since, the frequently used label is a fluorescent label, the signal from the label must be able to be distinguished from the autofluorescent signal. There is the problem of how to obtain a reliable fluorescent signal, in effect compromising maximizing the signal from the detectable label while minimizing the background signal.
In addition, the channel walls are not orthogonal to the cover plate, so that the depth of the irradiation will vary, depending upon the site of entry of the excitation beam into the channel. Where the excitation beam encounters the wall, the signal is degraded due to the reduced number of fluorophores which are excited and the excitation of the fluorophores in the wall. Therefore, precise positioning of the excitation beam in the channel is necessary for reproducible and accurate results.
2. Brief Description of Related Art
A number of patents have been published describing systems for detecting fluorescent signals in capillary arrays, such as U.S. Pat. Nos. 5,296,703 and 5,730,850, as well as WO98/49543.
An optical fluorescence detection system is provided for use with microfluidic arrays. The detection and orientation system comprises an optical train for receiving and processing light from a source of light and directing the light onto a microfluidic channel in a solid substrate. The optical train is moved across the surface of the solid substrate, crossing the channel and receiving the light emanating from the solid substrate. The optical train directs and processes the light from the solid substrate surface and directs the light to a detector. The signal from the detector is received by a data analyzer, which analyzes the signals and directs the optical train to the center of the channel in relation to the observed signals from the bulk material of the solid substrate, the edges of the channel and from the channel. Fluorescent components in the channel are detected by the fluorescence produced by the excitation light, where the emitted light is processed by the optical train and analyzed for the presence of fluorescence in the channel resulting from the fluorescent components in the channel, correcting for any fluorescence from the solid substrate.
The optical fluorescence detection system employs a plurality of miniaturized confocal microscope systems aligned in orientation with a plurality of channels of a microfluidic array. The systems are mounted on a movable support for alignment with sets of channels. The supports may be mounted on a carriage for alignment with different sets of channels. An irradiation unit comprises a source of light and processing means, such as lenses, dichroic mirrors, filters, gratings or the like, to reject light outside the wavelength range of interest. A single light source may be used and the beam split into a plurality of optical fibers for individual distribution of beamlets for channel irradiation. Similarly, the individual signals from each of the channels is directed by individual optical fibers to a common detector. Alternatively, individual light sources may be used for each confocal microscope system, such as LEDs or laser diodes.
The methodology allows for accurate, reproducible determination of a fluorescent signal from each of the channels. In order to achieve the desired sensitivity for detection, the center of each channel is determined, either when the channel is empty (air) or when a liquid is present, usually containing a fluorescent dye. Depending upon the degree of autofluorescence of the microfluidic array substrate, the optical system may look at fluorescent light, where there is sufficient autofluorescence to provide a detectable signal or scattered light, usually where the autofluorescence is low. In the case of scattered light, one would be detecting a different wavelength from the light, which would result from autofluorescence.
There are two different forms of delivering excitation: single mode fiber delivery or no fiber, where a laser and splitting must be done by discrete mirrors or a diffraction optical element; or multi-mode fiber delivery, where either a lamp or a laser may be used and splitting is done by homogenizing the laser or lamp light and then splitting using a multi-mode fiber array. The source of light will usually be a laser, generally being a laser producing a light beam having a wavelength in the range of about 250 to 800 nm, usually 488 nm, 532 nm or 633 nm.
Depending upon the source of light, such as a laser, a filter may be used to attenuate the intensity of the light to minimize photobleaching and photodestruction of the fluorescent labels. The light is then split into a plurality of rays or beamlets by a diffractive optical element, a combination of beam splitter elements, such as discrete mirrors, or other means, such as discrete beam splitters and fiber optic arrays. Each of the resulting beams is then directed to the individual confocal microscope associated with the channels. Either a single mode or multimode fiber may be employed, where one may use a multimode fiber optic array to split the illumination into N beamlets, where N is the number of optical trains to be illuminated. The fiber will generally have a diameter in the range of about 25 to 75 xcexcm, particularly about 50 xcexcm and a length in the range of about 1 to 1000 mm.
The confocal housing can be very compact, where the portion enclosing the optical train, usually in conjunction with other enclosed areas associated with the optical fibers and attachment to the orienting system, generally having a total volume of about 0.5 to 4xc3x97104 mm3, with a cross-section in the range of 200 to 2000 mm2 and a height in the range of about 25 to 200 mm. Each confocal microscope housing receives an individual light source optical fiber, with the fiber oriented such that the output face is normal to the optical axis of the housing and the light emerging is coincident with the optical axis. An optical system, usually involving a collimating lens and objective lens are positioned such that they focus the light from the fiber to a small spot. These lenses are usually aspherical with a single element. They are designed to be small, yet still offer diffraction limited performance.
Instead of having the optical fiber positioned at the optical axis, the chief ray from the optical fiber may be directed through a collimating lens which is outside the optical axis and collimates the light and directs the light to a dichroic mirror. The dichroic mirror directs the chief ray along the optical axis of the housing. The chief ray is focused by means of a lens with a high numerical aperture, generally in the range of about 0.25 to 0.75. The irradiation spot size will have a diameter of about 6-10 xcexcm, while the collection area will be about 200 to 600 xcexcm2. The excitation light will excite fluorophores present in the channel at the detection site and the fluorescent light emitted from the channel will be collected by the high numerical aperture lens. When a collimating lens is used, the light will be directed past the collimating lens. By proper positioning and design of the collimating lens photon losses due to obscuration by the collimating lens will be minimized. Where the dichroic mirror is employed, the mirror will be substantially transparent in the wavelength range of interest and the light beam focussed by the focussing lens will pass through the dichroic mirror. After passing through the dichroic mirror or past the collimating lens, the light beam will usually be filtered to remove light outside the wavelength range of interest and be refocused onto a plane that contains the entrance aperture or core of a multimode optical fiber. The emission fiber will have substantially the same dimensions as the excitation fiber. The aperture acts as the confocal aperture for the confocal assembly, although there are other ways to provide the confocal pinhole, such as avalanche photodiodes, and other detectors. The emission beam is received and directed by the emission optical fiber to a detector. Various detectors may be employed which have the appropriate sensitivity, such as photomultiplier tubes (PMTs), charged coupled detectors (CCDs), avalanche photodiodes, etc. The signal may then be processed to provide the level of emission obtained from the channel and relate this intensity to the amount of fluorophore in the channel. Since the amount of fluorophore will relate to an event of interest, it may serve to identify the nature of the sample.
In some situations one will be interested in signals coming from different fluorophores having different wavelength ranges. The emission light beam may be split into the number of different wavelengths of interest, using filters, dichroic mirrors, prisms and the like. Various commercial systems are available for this purpose, such as prisms, beam splitter mirrors, etc. The subject assembly with the fiber preserves the laser light source mode and profile and assures optimal focussing of the ray on the sample by the confocal microscope assembly.
The housings may be used individually, but will usually be used in combination to read a plurality of channels at detection sites. The individual housings are mounted on a support, which will usually be mobile to allow for the support to move and reorient the housings in relation to different sets of channels. For example, with 8 housings, one may read 8 channels, and by being able to move the support one may read different groups of 8 channels, so that with 12 readings, one could read the samples from a 96 assay plate pattern. By having 12 housings or more, usually not more than about 96 housings, one could read a large number of samples quickly, since an individual reading would take less than a few seconds and the movement of the support would be automated and the entire set of readings would be performed in less than about a minute. The support allows for movement of the housings, so as to orient the beam to substantially the center of the channel. Various methods may be used for controlling the movement of the housings, including mechanical, electromechanical, electromagnetic, and the like. The different methods may involve anchoring the housing to an arm mounted on a pivot rod, where the arm is restrained in one direction and urged in the opposite direction, a voice coil actuator, where the lever arm extends into the center of the coil. By using a control rocker arm which is cam operated, or a movable support which moves in a plane, the housing can be moved up to about a distance of about 10-1000xcexc, usually 500 xcexc, from a central point. Where the bulk material of the microfluidic chip is autofluorescence, the presence of the channel is determined by detecting the autofluorescence as one moves the illumination through a predetermined distance. With both autofluorescence and light scatter, where the bulk material is not significantly autofluorescent, there will be a channel signature as depicted to FIG. 9, showing the change in autofluorescent signal as the illumination traverses the channel.
The control arm is rigidly joined to the housing. The control arm is pivotally mounted on a bearing, so as to be able to move in a small arc about the channel. The arm can be actuated to scan the surface of the microfluidic chip about this arc, using the optical system for fluorescent detection to determine the site of the channel. Various actuators may be used for moving the arm and the housing, where the movement may be accelerated and decelerated as it passes through the arc. The observed autofluorescence is transmitted to the detector and the signals analyzed to determine the site of the channel. Once the borders of the channel have been determined, the housing and its optical axis may be oriented to be substantially above the center of the channel.
The length of the housing and lever arm will be relatively short, generally when measured from the axis of the bearing to the lens at the end of the housing adjacent to the microfluidic device, being in the range of 50 to 150 Movement of the housing will be controlled to at least steps of about 0.01xcexc, generally in the range of about 0.1 to 10xcexc.
Instead of using a mechanical arm, one may use various electromagnetic assemblies to control the movement of the housing in relation to an optical signal. By having opposing electromagnets or a single electromagnet with an opposing force, where the flux of the electromagnet is controlled by a computer, which relates the position of the housing to the change in signal as the housing traverses the channel area. Alternatively, one may use a motor and guide shaft for moving the housing, which allows the housing to traverse the channel area in a plane parallel to the surface of the chip.
Desirably one uses a single light source for a plurality of optical systems. The light from the single source is directed to a beam divider, such as a diffractive optical element or a system of beam splitters. Each of the beamlets is directed to an optical fiber which conducts the light to the optical system. While the light may be split into any number of rays, usually the total number of rays will not exceed 96, usually not exceed 64, more usually not exceed 32 and may be as few as 4, preferably from about 8 to 24. Each may be separated by an angle xcex8 in a linear array, but a two dimensional array may also be formed with the appropriate angle between rays. Each ray has similar propagation parameters as the input beam. In particular, the divergence, and transverse intensity profile are preserved. When the transverse intensity profile of the light source is the xe2x80x9cGaussianxe2x80x9d or TEM00, then each ray will preserve this profile. This profile permits optimal focussing. Each ray is propagated a sufficient distance to provide separation and a distinct position. The distance will generally be at least 1 mm, usually in the range of about 1 to 1,000 mm. Individual lenses, such as aspherical lenses, achromatic doublets, etc., focus each ray into a single mode optical fiber. Each fiber is connected to one of the confocal microscope assemblies which is associated with each channel.
The microfluidic array will be in a solid substrate, which may be an inflexible substrate or a flexible substrate, such as a film. For examples of microfluidic devices, see, for example, U.S. Pat. No. 5,750,015. If flexible, it will usually be supported and oriented in conjunction with a rigid support. The channels comprising the detection site will generally have a depth of about 10 to 200 xcexcm and a width at the opening of the channel in the range of about 1 to 500 xcexcm, usually 10 to 200 xcexcm. The channels may be parallel or in various arrays, where the inlet ports may be oriented in relation to a 96 or higher microtiter well plate, so that samples from the wells may be directly introduced into the port and microfluidic network. Depending on the purpose of the chip and the pattern of channels, whether the channels are straight, curved or tortuous, the chip may be only 1 or 2 cm long or 50 cm long, generally being from about 2 to 20 cm long, frequently 12.8 cm long. The width will vary with the number and pattern of channels, generally being at least about 1 cm, more usually at least about 2 cm and may be 50 cm wide, frequently about 8.5 cm wide. The chips will have inlet and outlet ports, usually reservoirs for buffer and waste which are connected to the channels and there may be additional channels connected to the main channel for transferring sample, reagents, etc., to the main channel. Electrodes will be provided for the channels, where the electrodes may be part of the chip, painted with electroconductive paint or metal plated on the chip, or electrodes may be provided for introduction into the reservoirs or channels by an external device. The spacing between the channels will usually be at least about 0.5 mm, more usually at least about 1 mm, at the detection site. Since the channels may take many courses and shapes, the distance between two adjacent channels may vary.
In order to make a series of determinations in the chip the chip is introduced into a module or group of modules, which will include the movable support. The chip will be indexed in relation to the support, so that the channels will be substantially oriented in relation to the optical axis of the associated housings. The module may also include electrodes or connectors to electrodes which are part of the chip, containers or other instrumentality, e.g. syringes, capillaries, etc., which can serve as sources of reagents, sample, and the like, which provide for fluid transfer through the ports in the chip, electrical connections between the fluorescent detectors and a data analysis system, and the like. The various modules are combined, so as to receive the chip and orient the chip in relation to the various components which interact with the chip. Indexing may be provided on the chip, so as to be locked in a predetermined position in relation to the module and the support. Prior to initiating operation in the channel, the housings are oriented in relation to the centers of the channels. Each of the housings is individually moved across the plane of the microfluidic chip intersecting the channel at the detection zone. Depending upon the level of autofluorescence of the composition of the substrate, autofluorescence or scattered light may be read. Where there is significant autofluorescence, autofluorescence or scattered light may be detected and read. Where the autofluorescence signal is low, scattered light will be read.
Where scattered light is being detected, the scatter will be different at the edges of the channel, as compared to the scatter from the channel. By observing the change in the scattered light, as the housing moves across the plane of the microfluidic chip, one can detect the transition from the edges of the channel to the channel and select the center as equally distant from the edges.
Once the housings are fixed in registry with the channel, the orientation process need not be repeated in relation to the channel and optical housing and numerous readings may be taken. One may then perform various operations, where a fluorophore label is brought to the detection site. The detection of the fluorophore label may be as a result of a competition assay, nucleic acid sequencing, immunoassays, etc.